Low-density Lipoprotein Receptor-related Protein 1 Deficiency Research Articles

The protective role of hippocampal LRP1 knockdown involves synaptic plasticity through the promoting microtubule dynamics and activation of Akt/GSK-3β pathway in depressive rats

BackgroundThe mechanism by which synaptic plasticity mediates the occurrence of depression is unknown. Low-density lipoprotein receptor-related protein 1 (LRP1) affects axon growth and neurogenesis in the brain, but its role in depressive-like behaviors is poorly understood. MethodsAdeno-associated virus-mediated small interfering RNA was injected into the bilateral hippocampus 14 days before chronic unpredicted mild stress (CUMS). Behavior performance was assessed for depressive-like behaviors. Western blot was conducted to detect levels of LRP1, neurogenesis-related proteins, synaptic markers, microtubule system molecules and Akt/GSK-3β signaling-related proteins. Immunohistochemical staining was performed for LRP1 protein, immunofluorescence staining was conducted to determine the Sox2 protein, Nissl's staining and transmission electron microscope staining were used to observe hippocampal morphological features. ResultsThe expression of hippocampal LRP1 was positively correlated with depressive-like behaviors. Treatment with iAAV-LRP1 exerted protective effects on depressive-like behaviors. LRP1 Knockdown relieved the inhibition of synaptic plasticity induced by CUMS. Expression of Sox2, GluR2 and SYP was significantly increased in iAAV-LRP1 CUMS rats. LRP1 knockdown reduced the p-tau (Ser262 and Thr404) and Acet-tubule levels in depressed rats. Finally, we found that LRP1 knockdown activated the PI3K/Akt pathway and inhibited GSK-3β signal transduction. LimitationsMore neurogenesis markers would be considered, and stereotactic injection into hippocampal DG region could be performed to investigate the effects of LRP1. ConclusionsThese findings indicated that hippocampal LRP1 deficiency in stressed rats plays an important protective role in depressive-like behavior by increasing synaptic plasticity mediated by microtubule dynamic and activating Akt/GSK-3β signaling pathway. Therefore, LRP1 may represent a potential therapeutic target for depression.

Heterozygous LRP1 deficiency causes developmental dysplasia of the hip by impairing triradiate chondrocytes differentiation due to inhibition of autophagy

Developmental dysplasia of the hip (DDH) is one of the most common congenital skeletal malformations; however, its etiology remains unclear. Here, we conducted whole-exome sequencing in eight DDH families followed by targeted sequencing of 68 sporadic DDH patients. We identified likely pathogenic variants in the LRP1 (low-density lipoprotein receptor-related protein 1) gene in two families and seven unrelated patients. All patients harboring the LRP1 variants presented a typical DDH phenotype. The heterozygous Lrp1 knockout (KO) mouse (Lrp1+/-) showed phenotypes recapitulating the human DDH phenotypes, indicating Lrp1 loss of function causes DDH. Lrp1 knockin mice with a missense variant corresponding to a human variant identified in DDH (Lrp1R1783W) also presented DDH phenotypes, which were milder in heterozygotes and severer in homozygotes than those of the Lrp1 KO mouse. The timing of triradiate cartilage development was brought forward 1 or 2 wk earlier in the LRP-deficient mice, which leads to malformation of the acetabulum and femoral head. Furthermore, Lrp1 deficiency caused a significant decrease of chondrogenic ability invitro. During the chondrogenic induction of mice bone marrow stem cells and ATDC5 (an inducible chondrogenic cell line), Lrp1 deficiency caused decreased autophagy levels with significant β-catenin up-regulation and suppression of chondrocyte marker genes. The expression of chondrocyte markers was rescued by PNU-74654 (a β-catenin antagonist) in an shRNA-Lrp1-expressed ATDC5 cell. Our study reveals a critical role of LRP1 in the etiology and pathogenesis of DDH, opening an avenue for its treatment.

Abstract 561: Micro-CT Visualizes Extensive Vascular Pathologies In Smooth Muscle Cell-specific LRP1 Deficient Mice

Objective: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional protein that is abundant in vascular smooth muscle cells (SMCs). SMC-specific deletion of LRP1 leads to vascular pathologies in the aorta and superior mesenteric artery (SMA) of mice. Although other vascular beds have not been explored. Micro-computed tomography (CT) enables scanning in a high spatial resolution (~20 μm). To determine the regional effects of SMC-specific LRP1 deficiency in mice, we performed detailed morphometric analyses of vascular pathologies using micro-CT. Approach and Results: Male and female wild-type and SMC-specific LRP1 deficient mice were monitored for aortic and SMA dilatations from 6 weeks through 40 weeks of age using ultrasonography. Progressive luminal dilatations of the ascending aorta were detected in both male and female SMC-specific LRP1 deficient mice, compared to their wild type littermates. Ultrasound was only able to detect the superior mesenteric artery within ~2 mm of the branch from the abdominal aorta. Therefore, ultrasonography is not optimal for monitoring the SMA. At the age of 40 weeks, mice were euthanized, perfused with saline and injected with Microfil silicone rubber through the left ventricle. Subsequently, each mouse was scanned by a Bruker SkyScan 1276 high-resolution desktop micro-CT and 3 dimensional images were generated. Aortic CT images showed profound dilatations of the entire aorta and its main branches, with more pronounced changes in the ascending thoracic and infrarenal aortic regions. Micro-CT also detected longer and tortuous aortic structure, compared to their wild type littermates. The most striking pathologies, including both dilation and tortuosity, were observed in the SMA and its branches with more depth and detailed insights, compared to ultrasonography. Conclusions: The ability to visualize the entire vasculature of mice using micro-CT technology demonstrated that SMC-specific deficiency of LRP1 has marked heterogeneity in development of vascular pathologies.

Heparan Sulfate Proteoglycans (HSPGs) Serve as the Mediator Between Monomeric Tau and Its Subsequent Intracellular ERK1/2 Pathway Activation.

The conversion of soluble tau protein to insoluble, hyperphosphorylated neurofibrillary tangles (NFTs) is a major hallmark leading to neuronal death observed in neurodegenerative tauopathies. Unlike NFTs, the involvement of monomeric tau in the progression of tau pathology has been less investigated. Using live-cell confocal microscopy and flow cytometry, we demonstrate that soluble 0N4R monomers were rapidly endocytosed by SH-SY5Y and C6 glioma cells via actin-dependent macropinocytosis. Further, cellular endocytosis of monomeric tau has been demonstrated to be HSPG-dependent, as shown in C6 glial cells with genetic knockouts of xylosyltransferase-1—a key enzyme in HSPG synthesis—with a reduced level of tau uptake. Tau internalization subsequently triggers ERK1/2 activation and therefore, the upregulation of IL-6 and IL-1β. The role of ERK1/2 in regulating the levels of pro-inflammatory gene transcripts was confirmed by inhibiting the MEK-ERK1/2 signaling pathway, which led to the attenuated IL-6 and IL-1β expressions but not that of TNF-α. Moreover, as a key regulator of tau internalization, LRP1 (low-density lipoprotein receptor-related protein 1) levels were downregulated in response to monomeric tau added to C6 cells, while it was upregulated in HSPG-deficient cells, suggesting that the involvement of LRP1 in tau uptake depends on the presence of HSPGs on the cell surface. The subsequent LRP1 knockdown experiment we performed shows that LRP1 deficiency leads to an attenuated propensity for tau uptake and further elevated IL-6 gene expression. Collectively, our data suggest that tau has multiple extracellular binding partners that mediate its internalization through distinct mechanisms. Additionally, this study demonstrates the important role of both HSPGs and LRP1 in regulating cellular immune responses to tau protein monomers, providing a novel target for alleviating the neuroinflammatory environment before the formation of neurofibrillary tangles.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12031-021-01943-2.

LRP1 regulates food intake and energy balance in GABAergic neurons independently of leptin action.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a member of LDL receptor family that plays a key role in systemic glucose and lipid homeostasis. LRP1 also regulates energy balance in the hypothalamus by mediating leptin's anorexigenic action, although the underlying neurocircuitry involved is still unclear. Because GABAergic neurons are a major mediator of hypothalamic leptin action, we studied the role of GABAergic LRP1 in energy balance and leptin action using mice lacking LRP1 in Vgat- or AgRP-expressing neurons (Vgat-Cre; LRP1loxP/loxP or AgRP-Cre; LRP1loxP/loxP). Here, we show that LRP1 deficiency in GABAergic neurons results in severe obesity in male and female mice fed a normal-chow diet. This effect is most likely due to increased food intake and decreased energy expenditure and locomotor activity. Increased adiposity in GABAergic neuron-specific LRP1-deficient mice is accompanied by hyperleptinemia and hyperinsulinemia. Insulin resistance and glucose intolerance in these mice are occurred without change in body weight. Importantly, LRP1 in GABAergic neurons is not required for leptin action, as evidenced by normal leptin's anorexigenic action and leptin-induced hypothalamic Stat3 phosphorylation. In contrast, LRP1 deficiency in AgRP neurons has no effect on adiposity and caloric intake. In conclusion, our data identify GABAergic neurons as a key neurocircuitry that underpins LRP1-dependent regulation of systemic energy balance and body-weight homeostasis. We further find that the GABAergic LRP1 signaling pathway modulates food intake and energy expenditure independently of leptin signaling and AgRP neurons.

Conditional Deletion of LRP1 Leads to Progressive Loss of Recombined NG2-Expressing Oligodendrocyte Precursor Cells in a Novel Mouse Model

The low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor, mediating endocytosis and activating intracellular signaling cascades. LRP1 is highly expressed in the central nervous system (CNS), especially in oligodendrocyte precursor cells (OPCs). Previous studies have suggested LRP1 as a regulator in early oligodendrocyte development, repair of chemically induced white matter lesions, and cholesterol homeostasis. To circumvent embryonic lethality observed in the case of global LRP1 deletion, we generated a new inducible conditional knockout (KO) mouse model, which enabled an NG2-restricted LRP1 deficiency (NG2-CreERT2ct2/wtxR26eGFPflox/floxxLRP1flox/flox). When characterizing our triple transgenic mouse model, we noticed a substantial and progressive loss of recombined LRP1-deficient cells in the oligodendrocyte lineage. On the other hand, we found comparable distributions and fractions of oligodendroglia within the Corpus callosum of the KO and control animals, indicating a compensation of these deficits. An initial study on experimental autoimmune encephalomyelitis (EAE) was performed in triple transgenic and control mice and the cell biology of oligodendrocytes obtained from the animals was studied in an in vitro myelination assay. Differences could be observed in these assays, which, however, did not achieve statistical significance, presumably because the majority of recombined LRP1-deficient cells has been replaced by non-recombined cells. Thus, the analysis of the role of LRP1 in EAE will require the induction of acute recombination in the context of the disease process. As LRP1 is necessary for the survival of OPCs in vivo, we assume that it will play an important role in myelin repair.

Low-density lipoprotein receptor–related protein-1 dysfunction synergizes with dietary cholesterol to accelerate steatohepatitis progression

Reduced low-density lipoprotein receptor-related protein-1 (LRP1) expression in the liver is associated with poor prognosis of liver cirrhosis and hepatocellular carcinoma. Previous studies have shown that hepatic LRP1 deficiency exacerbates palmitate-induced steatosis and toxicity in vitro and also promotes high-fat diet-induced hepatic insulin resistance and hepatic steatosis in vivo The current study examined the impact of liver-specific LRP1 deficiency on disease progression to steatohepatitis. hLrp1+/+ mice with normal LRP1 expression and hLrp1-/- mice with hepatocyte-specific LRP1 inactivation were fed a high-fat, high-cholesterol (HFHC) diet for 16 weeks. Plasma lipid levels and body weights were similar between both groups. However, the hLrp1-/- mice displayed significant increases in liver steatosis, inflammation, and fibrosis compared with the hLrp1+/+ mice. Hepatocyte cell size, liver weight, and cell death, as measured by serum alanine aminotransferase levels, were also significantly increased in hLrp1-/- mice. The accelerated liver pathology observed in HFHC-fed hLrp1-/- mice was associated with reduced expression of cholesterol excretion and bile acid synthesis genes, leading to elevated immune cell infiltration and inflammation. Additional in vitro studies revealed that cholesterol loading induced significantly higher expression of genes responsible for hepatic stellate cell activation and fibrosis in hLrp1-/- hepatocytes than in hLrp1+/+ hepatocytes. These results indicate that hepatic LRP1 deficiency accelerates liver disease progression by increasing hepatocyte death, thereby causing inflammation and increasing sensitivity to cholesterol-induced pro-fibrotic gene expression to promote steatohepatitis. Thus, LRP1 may be a genetic variable that dictates individual susceptibility to the effects of dietary cholesterol on liver diseases.

Deficiency of LRP1 in Mature Adipocytes Promotes Diet-Induced Inflammation and Atherosclerosis-Brief Report.

Mice with adipocyte-specific inactivation of low-density lipoprotein receptor-related protein-1 (LRP1) are resistant to diet-induced obesity and hyperglycemia because of compensatory thermogenic response by muscle. However, the physiological function of LRP1 in mature adipocytes and its role in cardiovascular disease modulation are unknown. This study compared perivascular adipose tissues (PVAT) from wild-type (adLrp1+/+) and adipocyte-specific LRP1 knockout (adLrp1-/-) mice in modulation of atherosclerosis progression. Analysis of adipose tissues from adLrp1+/+ and adLrp1-/- mice after Western diet feeding for 16 weeks revealed that, in comparison to adLrp1+/+ mice, the adipocytes in adLrp1-/- mice were smaller, but their adipose tissues were more inflamed with increased monocyte-macrophage infiltration and inflammatory gene expression. The transplantation of PVAT from chow-fed adLrp1+/+ and adLrp1-/- mice into the area surrounding the carotid arteries of Ldlr-/- mice before feeding the Western diet revealed a contributory role of PVAT toward hypercholesterolemia-induced atherosclerosis. Importantly, recipients of adLrp1-/- PVAT displayed a 3-fold increase in atherosclerosis compared with adLrp1+/+ PVAT recipients. The increased atherosclerosis invoked by LRP1-deficient PVAT was associated with elevated monocyte-macrophage infiltration and inflammatory cytokine expression in the transplanted fat. PVAT provide outside-in signals through the adventitia to modulate atherosclerotic lesion progression in response to hypercholesterolemia. Moreover, adipocytes with LRP1 deficiency are dysfunctional and more inflamed. This latter observation adds the adipose tissue to the list of anatomic sites where LRP1 expression is important to protect against diet-induced atherosclerosis.

Deficiency of the hepatokine selenoprotein P increases responsiveness to exercise in mice through upregulation of reactive oxygen species and AMP-activated protein kinase in muscle

Exercise has numerous health-promoting effects in humans; however, individual responsiveness to exercise with regard to endurance or metabolic health differs markedly. This 'exercise resistance' is considered to be congenital, with no evident acquired causative factors. Here we show that the anti-oxidative hepatokine selenoprotein P (SeP) causes exercise resistance through its muscle receptor low-density lipoprotein receptor-related protein 1 (LRP1). SeP-deficient mice showed a 'super-endurance' phenotype after exercise training, as well as enhanced reactive oxygen species (ROS) production, AMP-activated protein kinase (AMPK) phosphorylation and peroxisome proliferative activated receptor γ coactivator (Ppargc)-1α (also known as PGC-1α; encoded by Ppargc1a) expression in skeletal muscle. Supplementation with the anti-oxidant N-acetylcysteine (NAC) reduced ROS production and the endurance capacity in SeP-deficient mice. SeP treatment impaired hydrogen-peroxide-induced adaptations through LRP1 in cultured myotubes and suppressed exercise-induced AMPK phosphorylation and Ppargc1a gene expression in mouse skeletal muscle-effects which were blunted in mice with a muscle-specific LRP1 deficiency. Furthermore, we found that increased amounts of circulating SeP predicted the ineffectiveness of training on endurance capacity in humans. Our study suggests that inhibitors of the SeP-LRP1 axis may function as exercise-enhancing drugs to treat diseases associated with a sedentary lifestyle.

Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregulation depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes

The maintenance of sarcoplasmic reticulum Ca2+ ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca2+-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1α accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1α and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1α since HIF-1α overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1α in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure.

Abstract 463: Induction of Smooth Muscle-specific Deletion of Low-Density Lipoprotein Receptor-Related Protein 1 in Adult Mice Augments Angiotensin II Vascular Pathologies

Objective: Low-density lipoprotein receptor related protein 1 (LRP1) deletion in smooth muscle cells (SMCs) during embryonic development augments angiotensin (Ang) II induced aneurysm formation in the ascending aorta and superior mesenteric artery. However, these studies have the potential confounding issue of LRP1 deficiency causing structural changes to arteries that may be the basis for this augmented pathology. The purpose of this study was to determine whether AngII-induced vascular changes were influenced when LRP1 deletion occurred in adult mice. Methods and Results: To temporally delete LRP1 in SMCs, we used a tamoxifen (tam)-dependent Cre (ERT2) recombinase under the control of a mouse smooth muscle cell actin (SMA) gene. SMA-ERT2-Cre +/0 males containing the ROSA26 reporter were bred to LRP1 floxed female mice to develop SMA-ERT2-Cre 0/0 and +/0 LRP1 x ROSA26 mice. SMC-specific deficiency of LRP1 was induced in mature mice via an injection of an ERT2 ligand, tam (1.5 mg/kg for 5 consecutive days). Non- transgenic littermates were also injected with tam. Tam injections in mice expressing ERT2-Cre led to uniform staining of β-galactosidase in medial SMCs of the aorta and arteries. LRP1 was deleted effectively from SMCs in ERT2-Cre expressing mice as demonstrated by Western blotting of aortic medial extracts. AngII (1,000 ng/kg/min) was infused via mini-osmotic pumps for 28 days into tam-injected mice that were either ERT2-Cre 0/0 or +/0. Consistent with constitutive SMC deletion, SMC LRP1 deletion in mature mice had no effect on dilation of the suprarenal aorta, but augmented expansion of the thoracic aorta and superior mesenteric artery in both male and female mice. Conclusion: Consistent with constitutive deletion, SMC LRP1 deletion in mature mice resulted in augmentation of AngII-induced thoracic aortic and superior mesenteric arterial aneurysms. These results demonstrate that undefined ligands acting at LRP1 are responsible for site-specific AngII-induced vascular pathologies.

Neuronal LRP1 Regulates Glucose Metabolism and Insulin Signaling in the Brain

Alzheimer's disease (AD) is a neurological disorder characterized by profound memory loss and progressive dementia. Accumulating evidence suggests that Type 2 diabetes mellitus, a metabolic disorder characterized by insulin resistance and glucose intolerance, significantly increases the risk for developing AD. Whereas amyloid-β (Aβ) deposition and neurofibrillary tangles are major histological hallmarks of AD, impairment of cerebral glucose metabolism precedes these pathological changes during the early stage of AD and likely triggers or exacerbates AD pathology. However, the mechanisms linking disturbed insulin signaling/glucose metabolism and AD pathogenesis remain unclear. The low-density lipoprotein receptor-related protein 1 (LRP1), a major apolipoprotein E receptor, plays critical roles in lipoprotein metabolism, synaptic maintenance, and clearance of Aβ in the brain. Here, we demonstrate that LRP1 interacts with the insulin receptor β in the brain and regulates insulin signaling and glucose uptake. LRP1 deficiency in neurons leads to impaired insulin signaling as well as reduced levels of glucose transporters GLUT3 and GLUT4. Consequently, glucose uptake is reduced. By using an in vivo microdialysis technique sampling brain glucose concentration in freely moving mice, we further show that LRP1 deficiency in conditional knock-out mice resulted in glucose intolerance in the brain. We also found that hyperglycemia suppresses LRP1 expression, which further exacerbates insulin resistance, glucose intolerance, and AD pathology. As loss of LRP1 expression is seen in AD brains, our study provides novel insights into insulin resistance in AD. Our work also establishes new targets that can be explored for AD prevention or therapy.

Low-density lipoprotein receptor-related protein 1 mediates hypoxia-induced very low density lipoprotein-cholesteryl ester uptake and accumulation in cardiomyocytes

The myocardium accumulates intracellular lipids under ischaemic conditions, and myocardial fat deposition is closely associated with cardiac dysfunction. Our aims were to analyse the effect of hypoxia on low-density lipoprotein receptor-related protein 1 (LRP1) expression in neonatal rat ventricular myocytes (NRVM) and cardiac-derived HL-1 cells and the molecular mechanisms involved in this effect, to determine the role of LRP1 in the very low density lipoprotein (VLDL) uptake by hypoxic cardiomyocytes, and to study the effect of hypoxia on lipoprotein receptor expression and myocardial lipid profile in an in vivo porcine experimental model of acute myocardial infarction. Thin-layer chromatography after lipid extraction showed that VLDL exposure leads to cholesteryl ester (CE) and triglyceride (TG) accumulation in a dose-dependent manner and that hypoxic conditions further increased VLDL-derived intracellular lipid accumulation in HL-1 cells. Knockdown of LRP1 through lentiviral-mediated interfering RNA specifically prevented hypoxia-induced VLDL-CE internalization in HL-1 cells and NRVM. Lipopolysaccharide (LPS)-induced LRP1 overexpression specifically increased VLDL-CE accumulation in NRVM. In addition, using double-radiolabelled [(3)H]CE-[(14)C]TG-VLDL, we found that LRP1 deficiency specifically prevented hypoxia-induced VLDL-[(3)H]CE uptake. Finally, in an in vivo porcine model of infarcted myocardium, ischaemic areas exhibited LRP1 protein up-regulation and intramyocardial CE overaccumulation. Our results demonstrate that hypoxia increases LRP1 expression through HIF-1α and that LRP1 overexpression mediates hypoxia-induced VLDL-CE uptake and accumulation in cardiomyocytes.